Sep 24, 1990 - Cell suspensions of Serratia marcescens catalyzed the oxidation of aromatic aldehydes into the corresponding acids in high yield under mild ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1991, p. 1275-1276
0099-2240/91/041275-02$02.00/0
Vol. 57, No. 4
Oxidation of Aromatic Aldehydes by Serratia marcescens G. DE LA FUENTE,' F. PERESTELO,2* A. RODRiGUEZ-PEREZ,2 AND M. A. FALCON2 Instituto de Productos Naturales Organicos, Consejo Superior de Investigaciones Cientuficas,' and Departamento de Microbiologia y Biologia Celular, Universidad de La Laguna,2 La Laguna, Tenerife, Spain Received 24 September 1990/Accepted 18 January 1991 Cell suspensions of Serratia marcescens catalyzed the oxidation of aromatic aldehydes into the corresponding acids in high yield under mild conditions.
Recently, the chemical reactions performed by microorganisms or catalyzed by enzymes have been extensively studied from the viewpoint of synthetic organic chemistry (4, 12, 13). Aromatic aldehydes can be converted synthetically into carboxylic acids mainly by using permanganate, chromic acid, manganese dioxide, or silver oxide. In general, yields are high except when the aromatic nuclei are substituted by electron-releasing groups, which leads to ring cleavage, and when labile substrates are used in acid or basic conditions, if necessary (7). Despite the high dilution required, synthetic organic biological processes are a matter of increasing interest because of their well-known advantages (3, 13). In preceding studies (9, 10), we have reported production of vanillic acid, a valuable chemical (11), from vanillin by using growing and resting cells of Serratia marcescens. Several researchers reported such a microbial oxidation with Streptomyces viridosporus (2), Acinetobacter calcoaceticus (6), and three Desulfovibrio strains (14). However, our strain of S. marcescens supported a higher substrate concentration (9). Nevertheless, little is known about the applicability of such a technique for practical synthetic purposes. Here we report on an extension of this oxidation method to a variety of aromatic aldehydes. The following experiment is representative. Cells of S. marcescens were grown in basal mineral medium (8) supplemented with glucose (0.1% [wt/vol]) and vanillin (0.01% [wt/vol]). Cultures were grown aerobically at 28°C in 1-liter flasks containing 400 ml of medium on a reciprocal shaker (125 strokes per min), and bacterial growth was monitored by counting viable cells on nutrient agar (Oxoid) plates. After 28 h of incubation, at which time the specific activity of the aldehyde oxidase was maximal (9), the culture was divided into two 200-ml portions, and cells were harvested by centrifugation (5,000 x g, 4°C, 15 min). The cells from each portion were washed three times with 0.1 M phosphate buffer at pH 7 and then resuspended in the same buffer (100 ml, in 500-ml flasks) with aromatic substrates. The compounds used as substrates in this study and their concentrations (percent [weight per volume]) were transcinnamaldehyde, hydrocinnamaldehyde, and 2-nitrobenzaldehyde (0.05 and 0.1); iso-vanillin, salicylaldehyde, and 4-hydroxybenzaldehyde (0.05, 0.1, and 0.2); benzaldehyde and nicotinaldehyde (0.05, 0.1, 0.2, and 0.3); veratraldehyde, syringaldehyde, and iso-phthaldehyde (0.05, 0.1, 0.2, 0.3, and 0.4); and 3,4,5-trimethoxybenzaldehyde (0.05, 0.1, 0.2, 0.4, 0.8, 1.4, and 1.6). These compounds were supplied by Aldrich Chemical Co. *
Corresponding author.
Solutions containing the aldehydes were sterilized by filtration, and in the case of 3,4,5-trimethoxybenzaldehyde, the solution was warmed to 70°C in order to solubilize the substrate. The bioconversion media were incubated at 28°C on a reciprocal shaker, and the viability of the bacterial populations was monitored by counting viable cells on nutrient agar plates in order to determine the maximal nonbactericidal concentration of each substrate and to ensure that the cultures were not contaminated. Experiments at the maximal nonbactericidal concentration were done in duplicate, and control cultures of each substrate without bacteria were incubated under the same conditions as the inoculated cultures. Aliquots (1 ml) were withdrawn periodically, and reaction progress was monitored by UV-VIS spectroscopy and thinlayer chromatography (9). When the substrates disappeared, the cells were removed by centrifugation and washed three times with distilled water. Supernatants and wash fluids were combined, acidified to pH 2 to 3 with H2SO4 (20% [vol/vol]), except for nicotinic acid, and extracted to afford the corresponding acid (five 75-ml portions) with ethyl acetate (syringic, 3,4,5-trimethoxybenzoic, benzoic, 4-hydroxybenzoic, and iso-phthalic acids) or chloroform (veratric, iso-vanillic, salicylic, trans-cinnamic, 2-nitrobenzoic, hydrocinnamic, and nicotinic acids). Molar yields were determined gravimetrically and by UV spectroscopy, and the products were identified by characteristic melting point, mass spectrometry and 'H nuclear magnetic resonance (9). All aromatic aldehydes tested were oxidized into the corresponding carboxylic acid, which accumulated in the medium (Table 1). However, the highest concentration at which each substrate was transformed was different. The substrate concentration, limited by the bactericidal effect, was maximal with 3,4,5-trimethoxybenzaldehyde, of which up to 1.4 g could be used, even when the compound was not completely dissolved. In general, our S. marcescens strain tolerated higher concentrations of aromatic aldehydes than other bacteria (1, 2, 8). Although bacterial aromatic aldehyde oxidation is well known, such processes have only been studied extensively in Streptomyces viridosporus (2), Acinetobacter calcoaceticus (6), and three Desulfovibrio strains which did not oxidize salicylaldehyde (14). However, S. viridosporus catabolized benzaldehyde and 4-hydroxybenzaldehyde, as did A. calcoaceticus, in addition to salicylaldehyde and vanillin, indicating that enzymes of the ring cleavage pathway were probably not involved in our case. Vanillin, iso-phthaldehyde, salicylaldehyde, syringaldehyde and veratraldehyde at a 0.01% (wt/vol) concentration were oxidized into the corresponding acids in 2 to 7 days by S. viridosporus (2), and A. calcoaceticus did not oxidize 1275
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NOTES
TABLE 1. Oxidation of aromatic aldehydes into the corresponding acids by resting cells of S. marcescens Aldehyde UV Maximum nonbactericidal concna Molar yield of acid (%) Acid UV Time required absorption Time Aldehyde ~~~~~~~~~~~~~~~~~absorption reqiredn Aldehyde absorption (wt/vol) mM maximum version (h) SpectrophotoGravimetric % metric method method (nm)
nmaxmu
0.2 18.8 250 94 Benzaldehyde 0.1 255 8.2 99 Salicylaldehyde 0.1 8.2 94 4-Hydroxybenzaldehyde 283, 329 3.3 0.05 93 2-Nitrobenzaldehyde 223, 255 100 345 19.7b 0.3b Vanillin iso-Vanillin 277, 310 0.1 6.6 100 18.0 99 0.3 Veratraldehyde 230, 277 0.3 16.5 92 Syringaldehyde 306, 364 7.13 mmolb (3%) 96 1.4b,c (3%) 285 3,4,5-Trimethoxybenzaldehyde 3.7 (1%) ND" 0.05 (1%) Hydrocinnamaldehyde 258, 290 100 3.8 (0.5%) 0.05 (0.5%) 290 trans-Cinnamaldehyde 22.4 96 229 0.3 iso-Phthaldehyde 0.2 18.7 Nicotinaldehyde 232, 267 96 a Values in parentheses indicate the concentrations (vol/vol) of dimethyl sulfoxide used for solubilization. b Data are from reference 9. c
d
100 97 97 96 98 100 100 92 95 100 98 96 100
225 296 239 264
252, 285 250, 284 250, 283 260 251 260 268 204 262
45 10 4 4 96 4 230 230 100 3 3 68 72
Partially soluble. ND, Not determined.
aromatic aldehydes with a large substituent in the ortho position, such as 2-nitrobenzaldehyde (5). With S. marcescens the aldehyde conversions took place in 2 to 4 h at a 0.05% concentration, and 2-nitrobenzaldehyde was easily oxidized, indicating that the active enzymatic site did not seem to be restrictive. When 4-methoxyphenylacetone and vanillyl, 4-hydroxybenzyl, iso-vanillyl, veratryl, and 3,4,5-trimethoxybenzyl alcohols were used as the substrate at a 0.1% (wt/vol) concentration, oxidation to acid was only observed with the last three compounds at 1.7, 2.3, and 2.9% conversion, respectively, after 24 h. The low conversion observed with the alcohols was not due to contamination of the commercial samples with aldehydes; the purity was checked by thinlayer chromatography and gas chromatography. At that concentration, the corresponding aldehydes were completely converted in 4 to 6 h. On the other hand, 4-hydroxybenzyl, vanillyl, 2-hydroxybenzyl, and benzyl alcohols were catabolized by A. calcoaceticus (6). The high yields, mild neutral conditions, broad substrate specificity, and chemoselectivity make this bioconversion method a good alternative to the classical chemical methods for oxidation of aromatic aldehydes into carboxylic acids. This work was supported by grants 35-01688 (Gobiemo Au-
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